The nature and mechanism of action of signals that determine protein localization in cells are a primary focus of research in eukaryotic cell biology. The overall goal of this project is to elucidate in molecular detail the signal-dependent mechanisms by which transmembrane proteins are localized to the trans Golgi network (TGN) in eukaryotic cells, using localization of Kex2 protease in yeast as a model. Kex2p is the prototype of an important family of proprotein-processing proteases that function in late compartments of the secretory pathway in all eukaryotic cells. Thus this research is of significance not only for the knowledge it may provide about general principles of signal-dependent localization in the secretory pathway, but also because of the importance of understanding the cellular organization of proprotein processing, which is involved in a wide array of both normal and pathological phenomena in biology. During the current funding period, we have made significant progress in several areas. We have identified three genes, SOI1, SOI2 and SOI3, involved in localization of Kex2p and other transmembrane proteins to the yeast TGN. We have conducted detailed studies of the function of the SOI1 gene, which we found to encode a novel, conserved protein of 3144 residues that appears to function at both the TGN and the prevacuolar compartment (PVC, equivalent to the late endosome in animal cells) to promote the cycling of Kex2p and other TGN membrane proteins between the two compartments. We have identified two TGN localization signals (TLSs) in the Kex2p cytosolic tail. Soilp was shown to function through TLS1 to promote retrieval of Kex2p from the PVC and to function at the TGN to inhibit TLS2-dependent retention of Kex2p in the TGN. The work proposed here will expand on these results both to identify additional molecules involved in signal-dependent localization of TGN membrane proteins and to test aspects of the TGN-PVC cycling model. The specific aims of the project are: 1. to pursue a molecular, genetic and biochemical analysis of yeast Soilp and higher cell Soilp homologues; 2. to identify proteins that interact with Soilp using both genetic and biochemical approaches; 3. to clone and analyze the SOI2 and SOI3 genes; and 4. to identify and study additional genes involved in TGN localization through the isolation of multicopy suppressors of the Tyr713Ala mutation in the Kex2p TLS1 and identification of genes required for TLS2 function.